Isolation of skeletal muscle-derived mononuclear cells

 Protocol for the purification of quiescent satellite cells

1) Remove the muscle (TA, GC, Qu, Plantaris muscle, etc) from more than 10wks-old of C57BL/6 or KO mice.

2) Weigh the muscle mass. (If you spend many time to remove muscle, please leave them in cold PBS-containing dish.)

3) Please carefully remove tendon, adipose tissue, vessel etc in cold PBS.  After then mince the muscles on dish (not containing PBS).

4) Add 0.2% collagenase II (Worthington code: CLSS2) in DMEM (serum-free).  The volume is 3-4 times of muscle weight.  To treat the muscle with collagenase II, we use 10-20 ml beaker with a stir bar on magnetic stirrer (in 37 Cº incubator). 

5) After 1hr, please homogenize it with 18G-5ml syringe. If the suspension cannot pass through the 18G needle, mince the muscle mass more.  The suspension must pass completely at this time.

6) After additional treatment for 30 min, homogenize again at least 10 times.  Then, dilute the suspension with PBS (37 Cº) and homogenize 10 times. 

7) Muscle suspension is filtered through 100 µm mesh (BD Bioscience) and subsequently through 40 µm mesh (BD Bioscience). (My recommendation: Before filtrating, please dilute it with PBS  (50ml / approximately 1g muscle), and then perform the filtration. Recently we use only 40 µm mesh) 

8) Centrifuge it at 2000rpm for 5min or 1500rpm for 10 min. (My recommendation: We use one 50ml tube/ approximately 1g muscle). Remove supernatant.

9) Erythrocytes were eliminated by treatment with 0.8% NH4Cl in Tris-buffer solution for 2 min on ice. We use 1ml 0.8 NH4Cl / approximately 1g muscle. (Pink is the best color after the treatment. If the color is white, please shortent the time)

10) Add 50ml PBS-2%FCS (WB) and centrifuge 1500rpm x 5 min

11) After removing the supernatant, add 0.4ml PBS-2%FCS and perform cell count.

12) Add 2ul SM/C-2.6-biotin and incubate on ice for 30min

13) Add 50ml WB and centrifuge 1500rpm x 5 min

14) After removing the supernatant, add Streptavidin-APC or PE (I do not recommend Sta-FITC) and other labeled antibody (for example CD31-FITC, CD45-FITC, Sca-1-PE).  Incubate on ice for 30min.

15) Add 50ml WB and centrifuge 1500rpm x 5 min

16) After removing the supernatant, add 4-5ml WB per mice.

More information

When you collect mononuclear cells from regenerating muscle, we use Lympholyte to remove the debris. However, if you use Lymphocyte in the case of normal muscle, you will lose many cells in this treatment.  In order to purify myogenic cells from injured muscle (e.g. mdx mice), you must collect SM/C-2.6(+)CD31(-)CD45(-)Sca-1(-) fraction.

When you use Pax7-Rosa YFP mice, please skip 11)-15).

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